Protozoan parasites are a major cause of gastrointestinal disease. Within the last decade, the protozoa Cryptosporidium and Giardia have been increasingly associated with waterborne outbreaks of acute diarrhea. Cryptosporidium parvum is of particular concern because no known treatment of the illness is available at present. Moreover, in the immunocompromised host, a C. parvum infection can lead to prolonged severe diarrhea, malnutrition, wasting, and death.
Cryptosporidium is an enteric coccidia, which has a multi-staged life cycle one to eight days in duration. The oocyst contains four sporozoites which, during normal infection, are released in the presence of bile salts and proteases. The sporozoites attach and penetrate intestinal epithelial cells. Once inside, they develop into a rounded trophozoite in the area between the cytoplasmic membrane and the cytoplasm. Through asexual reproduction, the trophozoite (a type I meront) forms up to eight merozoites. The merozoites may then develop into a type II meront, which, by asexual reproduction, forms four merozoites. The second generation merozoites may develop into male (microgamont) or female (macrogamont) forms. The male form may lead to the sexual phase of the Cryptosporidium life cycle which culminates, in vivo, in the production of the environmentally resistant oocysts. These hardy structures possess a thick, double-layered protective cell wall which is resistant to most disinfectants, chlorine concentrations generally present in municipal water supplies, and temperatures between xe2x88x924xc2x0 C. and 42xc2x0 C.
Cryptosporidium is prevalent in most vertebrate groups. Domestic animals, such as rodents, kittens, puppies, and calves may constitute an important reservoir of the human Cryptosporidium. However, disease outbreaks in day-care centers, hospitals and urban family groups indicate that most human infections are transmitted person-to-person rather than via a zoonotic route. Since oocysts are found almost exclusively in stool, the transmission is undoubtedly fecal-oral. Moreover, the recovery of oocysts from both surface and drinking water suggests that indirect transmission via water is not uncommon.
Quantitative studies on the infectious dose for humans are currently limited. One study found that, in healthy volunteers, the infectious dose (ID50) is 132 oocysts, with as few as 30 oocysts causing infection in 20% of individuals tested (DuPont et al., 1995). However, the ID50 could be lower, such as one to ten oocysts, in more susceptible individuals.
Indeed, Cryptosporidium has been documented as a major cause of waterborne illness on numerous occasions. The largest outbreak occurred during the spring of 1993 in Milwaukee, Wis., resulting in approximately 400,000 illnesses and 100 deaths (MacKenzie et al., 1994).
Over the last 10 years, Cryptosporidium oocysts have been found in 9.1 to 100% of surface waters tested at concentrations ranging from 0.003 to 1,920 oocysts per liter. Oocysts were also detected in 27% and 17% of finished water samples in two multi-state surveys.
These studies, surveys, and documented outbreaks clearly indicate that infectious Cryptosporidium may be found in source water and the efficiency of conventional water treatment needs to be closely monitored. Indeed, the occurrence of the causative agents Cryptosporidium parvum and Giardia lamblia in water supplies has become a critical issue for the water industry.
The current techniques for isolating Cryptosporidium and Giardia from water involve filtration and centrifugation to concentrate and purify oocysts and cysts, respectively, followed by immunofluorescence microscopy. Objects with the correct shape, dimensions, and fluorescence are confirmed by observation of internal structures using differential interference contrast microscopy. The limitations of these procedures include loss of oocysts or cysts during isolation, resulting in recovery efficiencies ranging from 100 percent to less than one percent for Cryptosporidium. Moreover, the immunofluorescent assay (IFA) method cannot distinguish viable and potentially infective from non-viable or non-infective oocysts and cysts. Additional limitations of IFA include nonspecific antibody binding and cross-reactive antibody binding among human and animal infective species of Cryptosporidium or Giardia.
For the foregoing reasons, there is a need for an alternative method of detecting Cryptosporidium and Giardia pathogens that is rapid, sensitive, and specific. Moreover, the alternative method would be able to determine if Cryptosporidium oocysts are viable and infective.
The present invention includes a method for selectively detecting the presence of C. parvum organisms in a sample potentially containing C. parvum organisms and other Cryptosporidium organisms. The method comprises, first, selectively amplifying at least a portion of C. parvum HSP70 polynucleotide present in the sample using a primer, and then, detecting the presence of any amplified polynucleotide formed. The presence of amplified polynucleotide indicates the presence of C. parvum organisms in the sample. The method can additionally comprise recovering C. parvum oocysts from the sample, prior to amplifying the C. parvum polynucleotide. Further, the method can additionally comprise extracting C. parvum DNA from the recovered oocysts, prior to amplifying the C. parvum polynucleotide.
In a preferred embodiment, the primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In a preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10.
The present invention also includes a method for selectively detecting the presence of C. parvum organisms and for detecting the presence of G. lamblia organisms, simultaneously, in a sample potentially containing C. parvum organisms and G. lamblia organisms, and where the sample also potentially contains other Cryptosporidium species organisms. The method comprises, first, amplifying at least a portion of the G. lamblia HSP polynucleotide present in the sample using a first primer, and selectively amplifying at least a portion of the C. parvum HSP70 polynucleotide-present in the sample using a second primer, and then detecting the presence of any amplified polynucleotide formed. The presence of amplified C. parvum HSP70 polynucleotide indicates the presence of C. parvum organisms in the sample, and the presence of amplified G. lamblia HSP polynucleotide indicates the presence of G. lamblia organisms in the sample. The method can additionally comprise recovering C. parvum oocysts or G. lamblia cysts from the sample, prior to amplifying the C. parvum polynucleotide. Further, the method can additionally comprise extracting C. parvum DNA from the recovered oocysts or G. lamblia DNA from the recovered cysts prior to amplifying the C. parvum polynucleotide or the G. lamblia polynucleotide.
In a preferred embodiment, the first primer has a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, and the second primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In a preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, the complement of SEQ ID NO:9, the complement of SEQ ID NO:10 and the complement of SEQ ID NO:11.
The present invention further includes a method for selectively detecting viable C. parvum organisms in a sample potentially containing viable C. parvum organisms and other Cryptosporidium organisms. The method comprises, first, inducing mRNA transcription of C. parvum heat shock protein 70 (HSP70) DNA, and then, producing a C. parvum HSP70 polynucleotide, such as at least one copy of the mRNA or cDNA, from at least a portion of the C. parvum HSP70 mRNA utilizing a primer. Next, at least a portion of the C. parvum HSP70 polynucleotide is selectively amplified and the presence of any amplified polynucleotide formed is detected. The presence of amplified polynucleotide indicates the presence of viable C. parvum organisms in the sample. The method can additionally comprise recovering C. parvum oocysts from the sample, prior to amplifying the C. parvum polynucleotide. Further, the method can additionally comprise extracting C. parvum DNA from the recovered oocysts, prior to amplifying the C. parvum polynucleotide.
In a preferred embodiment, the primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In a preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10.
The present invention also includes a method for selectively detecting viable C. parvum organisms and for detecting viable G. lamblia organisms, simultaneously, in a sample potentially containing C. parvum organisms and G. lamblia organisms, and where the sample also potentially contains other Cryptosporidium species organisms. The method comprises, first, inducing mRNA transcription of C. parvum heat shock protein 70 (HSP70) DNA, and inducing mRNA transcription of a G. lamblia heat shock protein DNA, and then, producing a G. lamblia HSP polynucleotide, such as at least one copy of the mRNA or cDNA, from at least a portion of the G. lamblia HSP mRNA utilizing a first primer, and selectively producing a C. parvum HSP70 polynucleotide, such as at least one copy of the mRNA or cDNA, from at least a portion of the C. parvum HSP70 mRNA utilizing a second primer. Next, at least a portion of the G. lamblia HSP polynucleotide produced is amplified and at least a portion of the C. parvum HSP70 polynucleotide produced is amplified and the presence of any amplified polynucleotide formed is 25 detected. The presence of amplified C. parvum HSP70 polynucleotide indicates the presence of viable C. parvum organisms in the sample, and the presence of amplified G. lamblia HSP polynucleotide indicates the presence of viable G. lamblia organisms in the sample. The method can additionally comprise recovering C. parvum oocysts or G. lamblia cysts from the sample, prior to amplifying the C. parvum polynucleotide. Further, the method can additionally comprise extracting C. parvum DNA from the recovered oocysts or G. lamblia DNA from the recovered cysts prior to amplifying the C. parvum polynucleotide or the G. lamblia polynucleotide.
In a preferred embodiment, the first primer has a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, and the second primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In another preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, the complement of SEQ ID NO:9, the complement of SEQ ID NO:10 and the complement of SEQ ID NO:11.
The present invention additionally includes a method for selectively detecting infectious C. parvum organisms in a sample potentially containing infectious C. parvum organisms and other Cryptosporidium organisms. The method comprises, first, inoculating a cell culture with the sample, where the cell culture is susceptible to infection by infectious C. parvum organisms, and exposing the inoculated cell culture to conditions suitable to induce mRNA transcription of C. parvum heat shock protein 70 (HSP70) DNA. Next, a C. parvum HSP70 polynucleotide, such as at least one copy of the mRNA or cDNA, is produced from at least a portion of the C. parvum HSP70 mRNA utilizing a first primer and the C. parvum HSP70 polynucleotide produced is selectively amplified. Then, the presence of any amplified polynucleotide formed is detected. The presence of amplified polynucleotide indicates the presence of infectious C. parvum organisms in the sample. In one embodiment, the cell culture comprises a number of cells, the sample comprises a number of infective oocysts, and the number of cells exceeds the number of infective oocysts.
In a preferred embodiment, the first primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction. Further, the amplification can be performed in-situ.
In another preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10. Further, the detecting can be performed in-situ.
In a particularly preferred embodiment, the method additionally comprises detecting viable G. lamblia organisms in the sample, simultaneously with selectively detecting infectious C. parvum. In this embodiment, the method additionally comprises inducing mRNA transcription of mRNA transcription of a G. lamblia heat shock protein DNA. Then, a G. lamblia HSP polynucleotide, such as at least one copy of the mRNA or cDNA, is produced from at least a portion of the G. lamblia HSP mRNA utilizing a second primer and at least a portion of the G. lamblia HSP polynucleotide produced is amplified. The presence of any amplified polynucleotide formed is detected and the presence of amplified G. lamblia HSP polynucleotide indicates the presence of viable G. lamblia organisms in the sample. The method can additionally comprise recovering G. lamblia cysts from the sample, prior to amplifying the G. lamblia HSP polynucleotide.
In a preferred embodiment, the second primer has a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase B chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In another preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of consisting of SEQ ID NO:11 and the complement of SEQ ID NO:11.
The present invention also includes a method for selectively detecting infectious C. parvum organisms in a sample potentially containing infectious C. parvum organisms and other Cryptosporidium organisms. The method comprises, first, inoculating a cell culture with the sample, where the cell culture is susceptible to infection by infectious C. parvum organisms. Then, the inoculated cell culture is exposed to conditions suitable to induce mRNA transcription of C. parvum heat shock protein 70 (HSP70) DNA. Next, the presence of transcribed mRNA is selectively detected. The presence of amplified polynucleotide detected indicates the presence of infectious C. parvum organisms in the sample. The detection can be performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe, such as a DNA or PNA probe has a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10. Further, the detecting can be performed in-situ. Finally, the cell culture can comprise a number of cells, the sample comprises a number of infective oocysts, and the number of cells can exceed the number of infective oocysts.
The present invention also includes a kit for selectively detecting C. parvum. The kit comprises a first primer and a second primer for amplification of a portion of C. parvum HSP70 mRNA that is specific for C. parvum, and a PNA probe for detection of the amplified portion of C. parvum HSP70 polynucleotide. The first primer or the second primer can have a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The probe can have a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10.
The present invention further includes a kit for detecting C. parvum and G. lamblia. The kit comprises a first primer and a second primer for amplification of a portion of C. parvum HSP70 mRNA that is specific C. parvum, a third primer and a fourth primer for amplification of a target sequence of G. lamblia, and a PNA probe for detection of the amplified portion of C. parvum HSP70 polynucleotide. The kit can further comprise a PNA probe for detection of the amplified portion of the target sequence from G. lamblia. The first primer or the second primer can have a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The third primer or the fourth primer can have a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8. Further, the probe can have a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, the complement of SEQ ID NO:9, the complement of SEQ ID NO:10 and the complement of SEQ ID NO:11.